Pancreatic ductal adenocarcinoma (PDA) is characterized by rapid progression, early metastasis, and a limited response to chemotherapy and radiotherapy. The dismal prognosis associated with PDA is driving a search for specific molecular pathways that contribute to its invasive phenotype. Most studies performed to date on PDA have focused on the neoplastic cells of the tumor. However, it is now clear that the stroma surrounding the cancer cells plays a critical role in tumor growth and invasion. Tumor-associated fibroblasts (TAFs), the major cell type in the stroma, are believed to promote the motility of tumor cells to metastasize by contracting the extracellular matrix (ECM) and migrating through the matrix, thus creating channels that facilitate tumor cell migration. Conversely, neoplastic cells secrete cytokines and growth factors (such as TGF2 and PDGF) that strongly influence TAF behavior, triggering transdifferentiation to an activated phenotype. Recently published results have shown that the actin-binding protein palladin is dramatically upregulated in TAFs and thus may contribute to the acquisition of the activated TAF phenotype. In addition, we have generated novel preliminary results showing that activation of TAFs in vivo with phorbol ester-stimulation induces these cells to form invadopodia, which are actin-rich, matrix- degrading structures that have been identified previously in multiple types of invasive tumor cells. Our results demonstrate that (1) palladin is a component of invadopodia in TAFs, (2) palladin expression is required for invadopodium assembly, (3) the small GTPase Cdc42 is also required for invadopodium assembly, and (4) palladin contributes to the activation of the Cdc42. Together, these results suggest that TAFs may utilize palladin-dependent pathways to induce invadopodia formation, leading to ECM remodeling, and thus to enhanced metastasis of tumor cells. Our working hypothesis is that palladin expression plays an essential role in the assembly of invadopodia in TAFs by regulating the assembly of the actin cytoskeleton and the activity of the small GTPase Cdc42. To further define palladin's molecular function in TAFs, our specific aims are (1) to identify the palladin-dependent molecular pathways required for invadopodia formation using a mutagenesis approach of palladin's known domains and molecular interaction sites;(2) to identify and characterize the regulators of Cdc42 activity during invadopodia formation downstream of palladin. In pursuit of Aim 2, we will use pull down assays, followed by mass spectrometry analysis, to identify activators (GEFs) and inhibitors (GAPs) of GTPases in palladin-expressing and palladin-knockdown cells. Our overall objective is to identify novel molecular pathways that will form the basis for future research aimed at developing targeted therapies to control the metastasis-promoting behaviors of TAFs. PUBLIC HEALTH RELEVANCE: Pancreatic adenocarcinoma is an exceptionally lethal cancer characterized by rapid progression and early metastasis, and it is now clear that tumor-associated fibroblasts (TAFs), the major cell type in the tumor stroma, promote the metastasis of tumor cells by contracting the extracellular matrix and creating channels that facilitate tumor cell migration. Recently published results have shown that the actin-binding protein palladin is dramatically up-regulated in TAFs and thus may contribute to the acquisition of the invasive-promoting activity of TAFs. The proposed experiments will test the idea that TAFs may utilize palladin-dependent pathways to induce formation of invasive structures called invadopodia, leading to matrix remodeling, and thus to enhanced metastasis of tumor cells.